JP4865705B2 - Wheat having novel starch and method for producing the same - Google Patents

Description

  The present invention relates to wheat that does not express granular starch synthase and starch synthase type II protein in the endosperm of wheat.

Starch is a mixture of two components: linear amylose in which glucose is linked by α-1,4 bonds and amylopectin having a branched structure via α-1,6 bonds. These components are synthesized by the action of various enzymes and accumulate in the endosperm portion of seeds in cereals. Amylose is known to be synthesized mainly by the granule-binding starch synthase encoded by the granular starch synthase gene. On the other hand, amylopectin is synthesized by the action of multiple enzymes. These include (soluble) starch synthase type I, (soluble) starch synthase type II, (soluble) starch synthase type III, branching enzyme, debranching enzyme and the like.
Starch is also stored in plants in the form of highly crystallized granules. By adding water to this and heating it, the starch granules gradually swell, and at a certain temperature (gelatinization peak temperature), the crystal structure collapses at once and becomes paste-like (gelatinization). Thereafter, by cooling, the gelatinized starch gradually increases in viscosity and gels (ages). Such characteristics and the content ratio of amylose and amylopectin are known to vary greatly depending on the plant species from which they are derived.
Starch is a storage material in plants and an important energy source for animals. When ingesting starch, not only the grain containing it is processed and used in foods, but also used as an additive for thickeners, water retention agents, gel formers, etc., utilizing the above properties. On the other hand, it has been used industrially as a raw material for pastes and films. In addition, there is a great demand for processed starches that have been chemically or physically modified. Starch occupies most of the weight of stored organs (seed and tubers), and if the starch characteristics change, the effect on the texture or processability of the above products using them is great. There is a high demand for the development of starch with various characteristics.

The characteristics of starch as described above vary greatly depending on the plant species. However, the diversity of starch within the same plant species is largely dependent on changes in physical properties due to differences in amylose content. For example, in the case of normal starch in wheat, amylose content is about 30%, but a low amylose line with about 20% content is known. Low amylose-based wheat starch is said to be better used as a powder for noodles such as udon than the usual type, and is widely cultivated commercially. Rice and corn were known to accumulate waxy starch with extremely low amylose content, but wheat was first bred with Nakamura et al. (Patent Document 1), which is unique compared to normal types. It is known that it has a good workability and texture. The amylose content is often discussed as one of the characteristics representing the properties of starch, but when viewed within the same plant species, the diversity other than the amylose content is low. Therefore, since the starch derived from wheat or wheat flour containing this is low in diversity and only becomes a uniform product, the market is already matured. For this reason, if wheat that accumulates starch with new characteristics can be developed, it will be possible to develop improved products that give different characteristics from the past, or development of new applications. Is eagerly desired.
As an example of the production of wheat having novel characteristics, Yamamori et al. Have reported that a wheat line lacking starch synthase type II, which is one of the enzymes that synthesize amylopectin branched chains, has been developed (Non-Patent Literature). 1). It has also been reported that amylose is accumulated in a high content in this wheat, but research on other characteristics has not been advanced and has not been put into practical use.

In wheat, amylose is synthesized by the granular starch synthase protein encoded by the granular starch synthase gene. The wheat chromosome, which is an allogeneic hexaploid, has three genomes, A, B, and D, which are homoeologous chromosomes. Normally, there are three granular starch synthase genes, and these genes express granular starch synthase (granular starch synthase-A1, granular starch synthase-B1, granular Starch synthase-D1). However, there are types in which proteins are not expressed due to mutations that occur in genomic DNA, and there are 8 combinations of expression including wild type. It is known that there is a significant difference in the amylose content depending on this pattern. A wheat line lacking one or two is a low amylose line, and a type lacking all three is mochi wheat. As a method for easily identifying this defect pattern, a method of directly analyzing a protein expressed in endosperm or a method of examining based on a genomic DNA sequence has been established (for example, see Patent Document 1 and Non-Patent Document 2). ).
On the other hand, starch synthase type II protein is known as one of the enzymes involved in branched chain synthesis of amylopectin, but is encoded by three starch synthase type II genes present on the 7A, 7B, and 7D chromosomes. (Starch synthase type II-A1, starch synthase type II-B1, starch synthase type II-D1). As for starch synthase type II protein, a method for identifying a defect pattern has already been developed by the present inventors. Although this method can be used to classify the expression patterns of starch synthase type II protein into eight patterns, the diversity of starch characteristics in each pattern has not been sufficiently investigated.

JP-A-6-125669 Yamamori et. Al., Theor. Appl. Genet (2000) 101: 21-29 Nakamura et. Al., (2002) Genome 45: 1150-1156

  Accordingly, an object of the present invention is to provide a wheat that accumulates starch with new characteristics by controlling the expression of the enzyme.

As a result of intensive research to obtain wheat with new characteristics, wheat with very high raw starch degradation rate and very low by mutating specific genes and controlling the expression of specific proteins Wheat with gelatinized starch viscosity could be obtained. That is, the present invention relates to (1) starch synthase type II-A1 protein encoded by the starch synthase type II-A1 gene of SEQ ID NO: 1, and (2) starch synthase type II-B1 gene of SEQ ID NO: 3. Encoded starch synthase type II-B1 protein, (3) starch synthase type II-D1 protein encoded by SEQ ID NO: 5, and (4) granular starch of SEQ ID NO: 7 Granular starch synthase-A1 protein encoded by the synthase-A1 gene, (5) granular starch synthase-B1 protein encoded by the granular starch synthase-B1 gene of SEQ ID NO: 9, and (6) A wheat that does not express all of the granular starch synthase-D1 protein encoded by the granular starch synthase-D1 gene of SEQ ID NO: 11 is provided.
Moreover, this invention provides the wheat which does not have all the enzyme activities which the said protein (1)-(6) has.
The present invention also provides wheat in which 30% by weight or more of the accumulated starch does not form starch granules having a particle size of 10 μm or more.
The present invention also provides wheat in which the ratio of the number of branched chains having a degree of polymerization of 3 to 5 is 1.5% or more of the branched chains having a degree of polymerization of up to 70 in the amylopectin branched chain.
The present invention also provides wheat in which the rate of degradation of raw starch by amylase is 80% or more.

The present invention also provides wheat that does not express one or more of the proteins (1) to (3) and does not express one or more of the proteins (4) to (6) (provided that the protein (2) , (4) and (5) alone are excluded).
Further, the present invention has one or more enzyme activities of the proteins (1) to (3) and one or more enzyme activities of the proteins (4) to (6). Wheat that does not have the enzyme activity (except for the wheat that does not have only the enzyme activity of the proteins (2), (4) and (5)).
The present invention also provides a wheat that does not express any two of the proteins (1) to (3) and does not express one or more of the proteins (4) to (6).
Further, the present invention does not have any two enzyme activities of the enzyme activities of the proteins (1) to (3) and one or more enzyme activities of the proteins (4) to (6). A wheat having no enzyme activity is provided.
The present invention also provides wheat that does not express two or more of the proteins (1) to (3) and does not express two or more of the proteins (4) to (6).
Moreover, this invention does not have two or more enzyme activities which the said protein (1) to (3) has, and two or more enzyme activities which the said proteins (4) to (6) have. A wheat having no enzyme activity is provided.
The present invention also provides a wheat that does not express any two of the proteins (1) to (3) and does not express all of the proteins (4) to (6).
Further, the present invention provides all enzymes having the enzyme activity of any one of the enzyme activities of the proteins (1) to (3) and the enzyme activities of the proteins (4) to (6). Provide wheat with no activity.

The present invention also provides a wheat that does not express two or more of the proteins (1) to (3), wherein the combination of expression of granular starch synthase protein is the same as that of the wheat, and the protein (1) Provided is a wheat having a reduced viscosity after gelatinization of starch as compared to wheat expressing all of (3).
The present invention also provides a wheat that does not express two or more of the proteins (1) to (3), wherein the combination of expression of granular starch synthase protein is the same as that of the wheat, and the protein (1) Provided is a wheat having improved aging resistance after gelatinization of starch as compared with wheat expressing all of (3).
The present invention also provides:
(7) a starch synthase type II-A1 gene mutant in which at least nucleotides at positions 124 to 412 are deleted and / or substituted in the starch synthase type II-A1 gene of SEQ ID NO: 1; A starch synthase type II-B1 gene variant in which one or more bases are inserted at least between positions 6145-6146 in the starch synthase type II-B1 gene; and (9) starch synthase II of SEQ ID NO: 5 A step of detecting one or more starch synthase type II-D1 gene mutants in which at least positions 2590 to 2652 are deleted in the type-D1 gene, and (4) a granular starch synthase of SEQ ID NO: 7 Granular starch synthase-A1 protein encoded by A1 gene, (5) Granular starch synthase-B1 protein encoded by SEQ ID NO: 9 and (6) SEQ ID NO: 11 Granules Encoded by the Granular Starch Synthase-D1 Gene There is provided a method for selecting a specific wheat, comprising the step of detecting one or more of proteolytic starch synthase-D1 protein.

The present invention also provides a wheat containing 0.1% by weight or more of glucose in a mature seed excluding an embryo.
The present invention also provides a wheat containing 0.1% by weight or more of maltose in a mature seed excluding an embryo.
The present invention also provides wheat containing 1% by weight or more of sucrose in mature seeds excluding embryos.
Here, the mature seeds are seeds at the time of “the head of the head has turned yellow and the grains have reached waxy hardness” (Reversion Complete Book Vol. 1 Wheat (Agricultural Welfare), p. 88) and seeds of germination. It means something without.
The present invention also provides wheat in which the ratio of the number of branched chains having a polymerization degree of 2 to 5 is 3% or more of the branched chains having a polymerization degree of 60 in the glucose branched chain of amylopectin.
Moreover, this invention provides the foodstuff containing the said wheat or the starch derived from the said wheat.
Moreover, this invention provides the industrial product containing the starch derived from the said wheat or the said wheat.
Moreover, this invention provides the product processed using the starch derived from the said wheat or the said wheat.

  The wheat of the present invention comprises (1) starch synthase type II-A1 protein encoded by the starch synthase type II-A1 gene of SEQ ID NO: 1, and (2) starch synthase type II-B1 gene of SEQ ID NO: 3. Encoded starch synthase type II-B1 protein, (3) starch synthase type II-D1 protein encoded by SEQ ID NO: 5, and (4) granular starch of SEQ ID NO: 7 Granular starch synthase-A1 protein encoded by the synthase-A1 gene, (5) granular starch synthase-B1 protein encoded by the granular starch synthase-B1 gene of SEQ ID NO: 9, and (6) The granular starch synthase-D1 protein encoded by the granular starch synthase-D1 gene of SEQ ID NO: 11 is not expressed at all.

As an example of starch synthase type II-A1 protein (1) encoded by the starch synthase type II-A1 gene of SEQ ID NO: 1, starch synthase type II-A1 represented by the sequence shown in SEQ ID NO: 2 Examples include a protein or starch synthase type II-A1 protein having a homology with the sequence of SEQ ID NO: 2 of 90% or more. Here, the homology calculation is performed using Genetyx (Genetics).
As an example of the starch synthase type II-B1 protein (2) encoded by the starch synthase type II-B1 gene of SEQ ID NO: 3, starch synthase type II-B1 represented by the sequence shown in SEQ ID NO: 4 Examples include a protein or starch synthase type II-B1 protein having a homology with the sequence shown in SEQ ID NO: 4 of 90% or more.
As an example of the starch synthase type II-D1 protein (3) encoded by the starch synthase type II-D1 gene of SEQ ID NO: 5, starch synthase type II-D1 represented by the sequence shown in SEQ ID NO: 6 Examples include a protein or starch synthase type II-D1 protein having a homology with the sequence of SEQ ID NO: 6 of 90% or more.
As an example of the granular starch synthase-A1 protein (4) encoded by the granular starch synthase-A1 gene of SEQ ID NO: 7, the granular starch synthase-A1 represented by the sequence of SEQ ID NO: 8 Examples thereof include granular starch synthase-A1 protein having a homology of 90% or more with the protein or the sequence shown in SEQ ID NO: 8.
As an example of the granular starch synthase-B1 protein (5) encoded by the granular starch synthase-B1 gene of SEQ ID NO: 9, the granular starch synthase-B1 represented by the sequence described in SEQ ID NO: 10 Examples thereof include a granular starch synthase-B1 protein having a homology of 90% or more with the protein or the sequence shown in SEQ ID NO: 10.
As an example of the granular starch synthase-D1 protein (6) encoded by the granular starch synthase-D1 gene of SEQ ID NO: 11, granular starch synthase-D1 represented by the sequence of SEQ ID NO: 12 Examples thereof include granular starch synthase-D1 protein having a homology of 90% or more with the protein or the sequence shown in SEQ ID NO: 12.

It can be confirmed that the proteins (1) to (6) are not expressed by detecting the presence or absence and mutation of the gene encoding the proteins (1) to (6). As a method for confirming that the proteins (1) to (6) are not expressed, any method known to those skilled in the art can be used as long as it can detect a mutation in the gene sequence. For example, PCR method etc. are mentioned.
The PCR method is not particularly limited, and various known improved methods can be used. For example, in addition to a primer pair and template (test) DNA, Tris-HCl, KCl, MgCl ₂ , Reagents such as dNTP and TaqDNA polymerase are mixed to obtain a PCR reaction solution. One cycle of PCR consists of three steps: heat denaturation, primer annealing, and DNA synthesis with DNA polymerase. Each step requires a different reaction temperature and reaction time, so the range is set to an appropriate range depending on the base sequence of the DNA region to be amplified and its length. Thermal cyclers for such operations are commercially available. The detection sensitivity can be further improved by examining suitable PCR conditions such as the concentration of TaqDNA polymerase and MgCl ₂ and the number of reaction cycles, or using nested PCR.
PCR reaction products can be identified by immunoreaction or any way, but they can be electrophoresed and, if necessary, a clear band in the electrophoretic image using positive and negative controls. Can be confirmed that the detection substance (granular starch synthase gene and starch synthase type II gene mutant wheat) is present in the test substance.

Any primer can be used as the primer used in PCR as long as it can detect mutations in the gene encoding the proteins (1) to (6).
As a variant of the starch synthase type II gene, the present inventors have (7) starch in which at least the bases at positions 124 to 412 are deleted and / or substituted in the starch synthase type II-A1 gene of SEQ ID NO: 1. Synthase type II-A1 gene variant (SEQ ID NO: 27), (8) Starch synthase type II-B1 gene of SEQ ID NO: 3, wherein one or more bases are inserted at least between positions 6145-6146 Synthase type II-B1 gene variant (SEQ ID NO: 28) and (9) Starch synthase type II of SEQ ID NO: 5-Starch synthase type II in which at least bases at positions 2590 to 2652 are deleted in the D1 gene- A D1 gene variant (SEQ ID NO: 29) was found.
Therefore, as a primer capable of detecting a mutation in the starch synthase type II-A1 gene of SEQ ID NO: 1, for example,
(I) a primer that hybridizes upstream from position 124 of the starch synthase type II-A1 gene region whose SEQ ID NO: 1 at its 3 ′ end, and starch synthase type II-A1 whose 5 ′ end is SEQ ID NO: 1 A combination with a primer that hybridizes downstream from the position 412 of the gene region,
(Ii) a primer that hybridizes in such a manner that its 3 ′ end spans the deletion portion 124 to 412 downstream of position 412 of the starch synthase type II-A1 gene region of SEQ ID NO: 1, and its 5 ′ end A combination with a primer that hybridizes downstream from position 412 of the starch synthase type II-A1 gene region of SEQ ID NO: 1, and (iii) the starch synthase type II-A1 gene region of which the 3 ′ end is SEQ ID NO: 1 And a primer hybridizing upstream of position 124 of the above and a 5 ′ end thereof hybridizing so that the deletion portion 124 to 412 is straddled upstream of position 124 of the starch synthase type II-A1 gene region of SEQ ID NO: 1. A combination with a soy primer is mentioned.
Furthermore, the primers (i), (ii), and (iii) are desirably designed so that the starch synthase type II-A1 gene region can be specifically detected. Specifically, it is a primer designed in a region that does not completely match with other genome-derived starch synthase type II gene regions (starch synthase type II-B1, starch synthase type II-D1).
Specifically, a combination of a primer containing the sequence described in any one of SEQ ID NOs: 13 or 14 and a primer containing the sequence described in any one of SEQ ID NOs: 15 to 17 can be mentioned.

Further, as a primer capable of detecting a mutation in the starch synthase type II-B1 gene of SEQ ID NO: 3, for example,
(I) a primer that hybridizes upstream from position 6145 of the starch synthase type II-B1 gene region whose 3 ′ end is SEQ ID NO: 3; and a starch synthase type II-B1 whose 5 ′ end is SEQ ID NO: 3 A combination with a primer that hybridizes downstream from the position 6146 of the gene region,
(Ii) a primer that hybridizes to a base inserted between positions 6145 to 6146 of the starch synthase type II-B1 gene region of SEQ ID NO: 3 at the 3 ′ end, and starch of SEQ ID NO: 3 at the 5 ′ end A combination with a primer that hybridizes downstream from position 6146 of the synthase type II-B1 gene region,
(Iii) a primer that hybridizes upstream of position 6145 of the starch synthase type II-B1 gene region whose 3 ′ end is SEQ ID NO: 3 and starch synthase type II-B1 whose 5 ′ end is SEQ ID NO: 3 A combination with a primer that hybridizes to a base inserted between positions 6145-6146 of the gene region, and (iv) positions 6145-6146 of the starch synthase type II-B1 gene region whose 3 ′ end is SEQ ID NO: 3 A primer that hybridizes to the base inserted between and a primer whose 5 ′ end hybridizes to the base inserted between positions 6145 to 6146 of the starch synthase type II-B1 gene region of SEQ ID NO: 3. The combination of is mentioned.
Furthermore, the primers (i), (ii), (iii), and (iv) are preferably designed so that the starch synthase type II-B1 gene region can be specifically detected. Specifically, it is a primer designed in a region that does not completely match with other genome-derived starch synthase type II gene regions (starch synthase type II-A1, starch synthase type II-D1).
Specifically, a combination of a primer including the sequence described in any one of SEQ ID NOs: 18 to 20 and a primer including the sequence described in any one of SEQ ID NOs: 21 to 23 can be mentioned.

Further, as a primer capable of detecting a mutation in the starch synthase type II-D1 gene of SEQ ID NO: 5, for example,
(I) a primer that hybridizes upstream of position 2590 of the starch synthase type II-D1 gene region whose 3 ′ end is SEQ ID NO: 5, and starch synthase type II-D1 whose 5 ′ end is SEQ ID NO: 5 A combination with a primer that hybridizes downstream from the position 2652 of the gene region,
(Ii) a primer that hybridizes in such a manner that its 3 ′ end straddles the deletion portion 2590 to 2652 downstream of position 2652 of the starch synthase type II-D1 gene region of SEQ ID NO: 5, and its 5 ′ end A combination with a primer that hybridizes downstream from position 2651 of the starch synthase type II-D1 gene region of SEQ ID NO: 5; and (iii) the starch synthase type II-D1 gene region of SEQ ID NO: 5 at its 3 ′ end A primer that hybridizes upstream of position 2590 of and a 5′-end of the starch synthase type II-D1 gene region of SEQ ID NO: 5 so that the deletion portion 2590 to 2652 is bridged upstream of position 2590 A combination with a soy primer is mentioned.
Furthermore, the primers (i), (ii), and (iii) are desirably designed so that the starch synthase type II-D1 gene region can be specifically detected. Specifically, it is a primer designed in a region that does not completely match with other genome-derived starch synthase type II gene regions (starch synthase type II-A1, starch synthase type II-B1).
Specifically, the combination of the primer containing the sequence as described in any one of sequence number 24 or 25 and the primer containing the sequence as described in any one of sequence number 26 is mentioned.

  Moreover, as a method of detecting the mutation of the gene encoding the proteins (4) to (6), the method described in Nakamura et. Al., (2002) Genome 45: 1150-1156 can be used.

  Furthermore, as a method for detecting mutations in the gene encoding the above proteins (1) to (6), in addition to the PCR method, LAMP method, NASBA method, LCR method, SDA method, RCR method, TMA method, RT-PCR mRNA Qualitative or quantitative methods. Any method can be used as long as it can detect mutations in this gene sequence, but the LAMP method can be used as an example, as reported by Notomi et al. (Notomi et. Al., Nucleic Acids Research (2000). 28 , No. 12, e63). If the sequence to be detected at this time is the sequence of (1), the primer may be designed using the region sandwiching positions 412 to 412 of SEQ ID NO: 13 as the detection region. If the sequence to be detected is the sequence of (2), a primer may be designed using a part of the sequence from position 6145 to 6146 of SEQ ID NO: 14 as a detection region. If the sequence to be detected is the sequence of (3), a primer may be designed with the region sandwiching 2590 to 2652 of SEQ ID NO: 15 as the detection region. In addition to the designed primer, template DNA solution necessary for the reaction, dNTP, BstDNApolymerase, and other reagents necessary for the reaction are added, and the reaction is performed at 65 ° C, and the product is detected by an appropriate method, and starch synthase II Detection of type gene mutant wheat or determination of wheat starch synthase type II gene type. In other methods, similar detection and determination can be performed by drawing up the detection system according to the manual.

Moreover, as a method for confirming that the proteins (1) to (3) are not expressed, it is described in a report by Yamamori et al. (Yamamori et. Al., Theor. Appl. Genet (2000) 101: 21-29). The method is mentioned.
Moreover, as a method for confirming that the proteins (4) to (6) are not expressed, the method described in JP-A-6-125669 can be used.

  In the wheat of the present invention described above, 30% by weight or more of the accumulated starch does not form starch granules having a particle size of 10 μm or more. The ratio of forming starch granules having a particle size of 10 μm or more can be calculated by observing a cross section of wheat seed endosperm using an electron microscope or an optical microscope and determining the ratio of particles of 10 μm or more in a certain area in a certain area. Or it is calculated | required by isolate | separating the fraction which does not form a starch granule, and the fraction of a starch granule, and measuring the dry weight in the refinement | purification process of starch. By reducing the ratio of forming starch granules having a particle size of 10 μm or more, digestion with amylolytic enzymes such as amylase and pullulanase is likely to occur, and easily digestible starch can be obtained. By using such starch, it is possible to provide a food that is easily digested and easily absorbed into the body. In addition, since starch forms a high-order structure called starch granules (crystallization), it does not completely gelatinize even when heated in boiling water. Since it is considered that no structure is formed, gelatinization may be achieved at a lower temperature for a shorter time, and the material is suitable for cooking at a lower temperature for a shorter time. If cooking at a low temperature for a short time is possible, there is an advantage that the influence of heating on other materials can be remarkably reduced or energy can be saved. Furthermore, since it does not form particles, it is suitable for use as a raw material for films and the like.

Furthermore, in the wheat of the present invention described above, the ratio of the number of glucose branched chains having a degree of polymerization of 3 to 5 in the amylopectin branched chain having a degree of polymerization of up to 70 is 1.5% or more. Preferably, it is 3% or more. In the wheat of the present invention described above, the ratio of the number of glucose branched chains having a degree of polymerization of 2 to 5 in the chain having a degree of polymerization of up to 60 is 3% or more. Preferably, it is 5% or more. By having such a chain length distribution, the structure as starch can be greatly changed. In fact, in the present invention, by using this type of starch, starch grains are not formed, and structural characteristics can be greatly changed. The sugar component that could not actually form starch granules is accumulated as a base material consisting of a smooth continuous layer. In the starch refining process, a gel-like layer is formed, which has a multifaceted effect on physical properties. Yes. Normally, almost no gel-like substance accumulates in the starch purification process, and it can be used as a raw material for industrial products such as films.
Moreover, since this gel-like substance has a very high water retention capacity, it can be expected to be used as a water retention agent.
Alternatively, it can be used as a surface coating agent.
Amylopectin is a macromolecule composed of α-1,4 bonds linked in a straight chain and branched by α-1,6 bonds, and its molecular weight is between amylopectin molecules. There is diversity in the number and length of branches. Therefore, the chain length distribution of the glucose branched chain of amylopectin shown in the present invention shows an average distribution in the whole amylopectin accumulated in seeds.

  Further, the wheat of the present invention described above has a decomposition rate of raw starch by amylase of 80% or more. Preferably, it is 90% or more. The improvement in the degradability in the state of raw starch without gelatinization by heat is considered that such starch is easily digested and absorbed without cooking. Alternatively, it can be used as a raw material for biodegradable plastics.

  Other wheat of the present invention includes (1) starch synthase type II-A1 protein encoded by SEQ ID NO: 1 starch synthase type II-A1 gene, and (2) starch synthase type II type SEQ ID NO: 3. -Starch synthase type II-B1 protein encoded by B1 gene, (3) Starch synthase type II-D1 protein encoded by SEQ ID NO: 5, (4) SEQ ID NO: 7 Granular starch synthase-A1 protein encoded by the granular starch synthase-A1 gene of (5) granular starch synthase-B1 protein encoded by the granular starch synthase-B1 gene of SEQ ID NO: 9, And (6) it does not have all the enzyme activities of the granular starch synthase-D1 protein encoded by the granular starch synthase-D1 gene of SEQ ID NO: 11.

As described above, the proteins (1) to (3) are generally synthesized from amylopectin branched chains (glucose polymers branched by α-1,6 bonds), particularly chains having a medium degree of polymerization (15 It is said to be involved in the synthesis of chains having a degree of polymerization of about 25 to 25. Therefore, the enzymatic activity of this protein is also considered to be the activity of recognizing and binding ADP-glucose as a substrate and amylopectin and binding glucose from ADP-glucose to the end of the amylopectin branch chain. On the other hand, the proteins (4) to (6) are considered to be basically involved in amylose synthesis. Therefore, it is considered that the enzyme activity is a function of recognizing and binding ADP-glucose and amylose as substrates, and adding glucose from ADP-glucose to the terminal of amylose during elongation.
In order to confirm the enzyme activity of the proteins (1) to (6) above, any commonly used method may be used. For example, this enzyme is purified from seeds, and [U-14C] ADP-glucose, glycogen or amylopectin as a substrate, and a component for adjusting reaction conditions are added to carry out the reaction. When the reaction is completed for a certain period of time, the enzyme is inactivated by heating to 100 ° C, and unreacted [U-14C] ADP-glucose is removed using an anion exchange column, which is then incorporated into glycogen or amylopectin. The amount of [U-14C] ADP-glucose is measured using a liquid scintillation counter. As an alternative method, acrylamide gel electrophoresis of protein fraction roughly purified from seeds or starch fraction (5-10 μg protein content) under the condition of SDS-PAGE excluding SDS and β-mercaptoethanol. I do. The gel after separation is immersed in a solution consisting of 50 mM glycine, 100 mM ammonium sulfate, 5 nM β-mercaptoethanol, 5 mM MgCl2, 0.5 mg / ml bovine serum albumin, 0.01 mg / ml glycogen or amylose, and 4 mM ADP-glucose. Leave for 12 hours from time. Thereafter, a method may be used in which staining is performed by adding a solution comprising 0.2% iodine and 0.02% potassium iodide to determine enzyme activity.
Further, as described above, a method for confirming the expression of the protein itself or a method for identifying a mutation on genomic DNA that causes loss of enzyme activity may be used. In the proteins (1) to (3), the mutation on the genomic DNA that causes the loss of enzyme activity is the recognition of ADP-glucose and amylopectin, which are enzyme substrates, the mutation at the binding site, or the non-mutation of glucose to amylopectin. Examples include a mutation at the active center site that transfers to the reducing end, or a mutation at the signal sequence site that exists at the N-terminus. In the proteins (4) to (6), the mutation on the genomic DNA that causes the loss of enzyme activity is the recognition of ADP-glucose and amylose which are enzyme substrates, the mutation at the binding site, or the non-change of glucose in amylose. Examples include mutation at the active center site that transfers to the reducing end. In particular, when mutagenesis treatment is performed by irradiation or administration of a mutagenic chemical substance, there is a possibility that a mutation accompanied by amino acid substitution of only one residue may occur. In such cases, since it is often difficult to identify by confirmation by SDS-PAGE, a technique for confirming mutation on DNA is suitable. The method for confirming the mutation on the DNA sequence is as described above.

  Other wheat of the present invention does not express one or more of the proteins (1) to (3) and does not express one or more of the proteins (4) to (5) (provided that the protein (2), except for wheat that does not simultaneously express (4) and (5) alone). These wheat accumulate starch with new properties compared to starch accumulated in conventional wheat. Examples of the new characteristics include modifications such as the viscosity of gelatinization liquid, gelatinization peak temperature, aging resistance, freeze-thaw resistance, and degradability by digestive enzymes. In particular, in the present invention, it has been found that the loss of one or more starch synthase type II proteins increases the degree of gelatinization compared to the type without loss, the viscosity of the gelatinization solution is low, and the degree of aging is low. . Moreover, the effect was found to change dramatically by simultaneously deleting one or more granular starch synthases. Starch accumulated in such wheat accumulates starch having a higher degree of gelatinization, starch having a lower viscosity, or starch having improved aging resistance than conventional ones when cooled after gelatinization. In particular, wheat that does not express one of the proteins (1) to (3) and does not express two of the proteins (4) to (6) expresses all of (1) to (3), and ( As compared with starch of wheat which does not express two of 4) to (6), aging resistance after gelatinization and freeze-thaw resistance are dramatically improved. Alternatively, wheat that does not express two of the proteins (1) to (3) and does not express all of the proteins (4) to (6) has a very low viscosity of the gelatinization solution. . For the confirmation of such wheat, a method of detecting the presence or absence and mutation of the gene encoding the protein (1) to (6) may be used, or a method of confirming the expression of the protein itself may be used. May be. Alternatively, a method may be used in which RNA expressed from the gene sequences described in SEQ ID NOs: 1 to 11 is purified and qualitatively or quantified.

Preferably, the wheat does not express any two of the proteins (1) to (3) and does not express one or more of the proteins (4) to (6). In these wheats, the viscosity after gelatinization is significantly lower than that of conventional wheat. This feature is particularly remarkable in wheat that does not express two of the proteins (1) to (3) and does not express all of the proteins (4) to (6).
Preferably, the wheat does not express two or more of the proteins (1) to (3) and does not express two or more of the proteins (4) to (6). In particular, these wheats have improved aging resistance after gelatinization compared to conventional wheat. This feature is particularly remarkable in wheat that does not express any two of the proteins (1) to (3) and does not express all of the proteins (4) to (6).

In addition, the other wheat of the present invention does not have one or more of the enzyme activities of the proteins (1) to (3) and the enzyme activities of the proteins (4) to (6). (Excluding wheat that does not have only the enzyme activity of proteins (2), (4) and (5)).
More preferably, it does not have two or more enzyme activities of the protein (1) to (3) and has two or more enzyme activities of the protein (4) to (6). It is a wheat that does not have.
More preferably, the protein (1) to (3) does not have any two enzyme activities, and the proteins (4) to (6) have one or more enzyme activities. It is a wheat with no activity.
More preferably, it does not have any two enzyme activities of the protein (1) to (3) and has all the enzyme activities of the proteins (4) to (6). It's a waste wheat.

Further, by preventing two or more of the proteins (1) to (3) from being expressed in wheat, the combination of expression of granular starch synthase protein is the same as that in the wheat, and protein (1) Compared with wheat that expresses all (3), the viscosity of starch after gelatinization can be reduced.
Further, by preventing two or more of the proteins (1) to (3) from being expressed in wheat, the combination of expression of granular starch synthase protein is the same as that in the wheat, and protein (1) Compared with wheat that expresses all of (3), the aging resistance after gelatinization of starch can be improved.

  From the above, specific wheat is detected by using the steps of detecting one or more of the gene variants (7) to (9) and detecting one or more of the proteins (4) to (6). Can be selected. Here, examples of the specific wheat include wheat that does not have all the proteins (1) to (6) above, such that the raw starch has a decomposition rate of 80% or more. Alternatively, among the proteins of (1) to (3) above, the protein of (1) and (2) is not expressed and the protein of (4) to (6) Examples include wheat that does not express at all.

Moreover, the wheat of this invention and the starch derived from the wheat of this invention can be used for a foodstuff. Examples of the food include foods usually using wheat flour (including whole grain flour) or starch. For example, it can be used as a bridge for bakery foods, noodles, confectionery, fried foods, grilled foods, kneaded foods, hamburgers and the like. Flour or starch obtained from this wheat may be used as it is, or may be used by mixing with other flour. It can also be used as a fermentation raw material for alcoholic beverages and a raw material for producing microorganisms such as amino acids and polysaccharides.
The bakery food made using wheat in the present invention includes, for example, bread such as bread, French bread, roll bread and sweet bread, fried bread such as yeast donut, steamed bread, pizza such as pizza pie, sponge cake, etc. Cakes such as baked confectionery such as cookies and biscuits. As wheat flour derived from the present invention used for producing these bakery foods, flour from which components such as bran have been removed through a normal milling process may be used, or whole grain flour that is not fractionated may be used. As the flour for bakery food for producing the bakery food, the flour obtained from the wheat of the present invention may be used as it is, but preferably mixed with other flour. Examples of the other flour include wheat flour such as strong flour, medium flour, and weak flour, or wheat-derived flour, rye flour, rice flour, starch, etc. that are not classified into these flours. The bakery food of the present invention is a flour obtained from the wheat of the present invention or a mixed flour with other flour, a chemical swelling agent such as yeast, baking soda, yeast food, salt, sugar, fats, eggs, dairy products, water, etc. These are generally produced by kneading a mixture of various auxiliary materials used in the manufacture of bakery foods to make a dough, which is expanded by fermentation or the like, or baked or fried as it is. In the production of the bakery food of the present invention, any of the commonly employed production methods, production devices, freezing methods, and freezing devices may be used. Moreover, additives, such as a vitamin and a mineral, can be added as needed.
The bakery food containing the wheat flour derived from the present invention produced as described above is sweet and has a unique flavor, aroma, and texture. Such a feature is also achieved when only the wheat-derived flour of the present invention is used. Preferably, the wheat-derived flour of the present invention contains 0.1 to 60 parts by mass, more preferably 0.5 to 50 parts by mass. It can be achieved by using it in combination with flour.
The wheat of the present invention and the starch derived from the wheat of the present invention can be used for industrial products. Examples of the industrial products include viscosity stabilizers, water retention agents, colloidal stabilizers, glues, and adhesives.
In addition, the wheat of the present invention and the starch derived from the wheat of the present invention can be used after being processed. For example, a method of producing dextrin by acid, alkali, or enzyme treatment and using it for an adhesive, fiber, film or the like can be mentioned. In addition, the water-soluble fraction of this dextrin can be used as a functional food excellent in intestinal action as an indigestible dextrin. Alternatively, starch esters may be formed by reacting with various inorganic and organic acids. In particular, starch phosphate reacted with phosphoric acid is useful as a thickener.

In addition, wheat that does not express all of proteins (1) to (6) has an increased sugar content as compared to normal wheat. Specifically, glucose is contained in 0.1% by weight or more in mature seeds excluding embryos. Preferably, 0.3% by weight or more of glucose is contained in the mature seed excluding the embryo. More preferably, the mature seed excluding the embryo contains 0.5% by weight or more of glucose. Further, maltose is contained in an amount of 0.1% by weight or more in the mature seed excluding the embryo. Preferably, 0.3% by weight or more of maltose is contained in the mature seed excluding the embryo. More preferably, maltose is contained in an amount of 0.5% by weight or more in the mature seed excluding the embryo. Moreover, sucrose is contained 1% by weight or more in the mature seed excluding the embryo. Preferably, 3% by weight or more of sucrose is contained in the mature seed excluding the embryo. More preferably, the mature seed excluding the embryo contains 5% by weight or more of sucrose.
As a method for measuring the content of glucose, maltose and sucrose in seeds, any method may be used as long as it is a method for extracting low molecular sugar from seeds and performing analysis. Methods for recovering low-molecular sugars from seeds include pulverization of seeds, extraction with water, extraction with 80% ethanol, and extraction with DMSO. Starch or polysaccharides are degraded by enzymes such as amylase during the operation. It is desirable to extract and measure under conditions where the action of amylase does not work, in order to clearly distinguish it from the sugars produced. In addition, since the present invention is a wheat that accumulates sugar at a high content in the wheat endosperm part, it is preferable to use a part obtained by removing the embryo from the seed grain as a sample for the measurement. As a method for identifying and quantifying low-molecular sugars contained in the extracted sample, any method such as a method using capillary electrophoresis, a method using HPLC, an enzyme, or a measurement method using a chemical reaction may be used.

  In the wheat of the present invention, the F1 generation obtained by crossing the above-mentioned wheat with other varieties may be further self-fertilized or backcrossed with one parental line to introduce other useful traits. included. For example, the above-mentioned wheat is crossed with a generally known highly disease-resistant variety, and the resulting F1 generation is backcrossed with a parent disease-highly resistant variety. Regarding the obtained next-generation individuals, individuals expressing granular starch synthase and starch synthase type II proteins in a desired combination are selected by the method described above. By further repeating backcrossing and selection for the selected individuals, desired starch characteristics can be imparted to varieties with high disease resistance. Alternatively, disease resistance can be imparted to the above-mentioned wheat by selecting the parent line to be backcrossed as the above-mentioned wheat and using an appropriate method. Other useful traits include, but are not limited to, gluten characteristics, lodging resistance, autumn sowing ability, spring sowing ability, high yield, low temperature tolerance, ear germination tolerance, and milling suitability.

1. Samples Wheat line granular starch synthase and starch synthase type II protein type developed according to the present invention are compared with standard lines (type (i)) or parent lines (type (ii) and type (iii)). ) And are shown in Table 1. The wheat line used as the parent line for the present invention is the wheat cultivar Mochi Otome (granular starch synthase deficient type, starch synthase type II protein is wild type) and foreign varieties grown at the Tohoku Agricultural Research Center. The lines were crossed and the F5 generation seeds lacking granular starch synthase were selected (type (ii)). As the other parent line, a starch synthase type II protein-deficient line grown in the same place was used. This strain is generally known as Kanto 79 (starch synthase type II-B1 deficient type) and foreign varieties Turkey 116 (starch synthase II type-D1 deficient type), Chosen57 (starch synthesis) Three varieties (enzyme type II-A1 deficient type) were sequentially crossed, and a line completely lacking starch synthase type II was selected (type (iii)).
Wheat cultivation and mating followed the standard method. Two varieties that became the parent lines were crossed to obtain the F1 generation. F2 or later generation is obtained by self-fertilization. From this, the desired wheat line is analyzed for the presence of granular starch synthase by two-dimensional electrophoresis and the starch synthase type II gene by PCR. Selection was made by identifying the type (wild type or mutant). The wheat line used as a comparative control was Norin 61 (type (i)), which is a cultivated and distributed variety.

The confirmation of the wild type and defective type of granular starch synthase was confirmed by two-dimensional electrophoresis of starch-binding protein. The method is as follows.
The embryos were removed from the mature seeds, crushed, and the powder was prepared through a 60 μm sieve. SDS buffer (0.1 M Tris-HCl (pH 6.8), 2.3% SDS, 5% β-mercaptoethanol, 10% glycerol) cooled at a rate of 1 mL per 20 mg of powder was added and homogenized for 2 minutes. After filtering this suspension, the filtrate was centrifuged at 15000 rpm for 5 minutes, the supernatant was removed, SDS buffer was added again, and the mixture was suspended and centrifuged. After repeating this operation three times, distilled water was added and the same operation was repeated twice, the supernatant was removed, and the recovered starch layer was naturally dried to obtain purified starch.
In the case of normal type starch, in the purification of the starch, after adding SDS buffer and filtering, most of the starch precipitates in the stage of centrifugation to form a starch grain layer, which is clearly the upper water layer. Are distinguished. In the type (vii) wheat of the present invention, what appears to be a starch granule layer precipitates very little at this stage, and a large amount of a layer composed of a cloudy amorphous gel-like substance is formed on the upper layer. Accumulated and a water layer is formed on the upper layer. After removing the aqueous layer, the gel-like layer, which is a sugar component, and the lowermost starch granule layer were combined and the subsequent operation was performed in the same manner as in the purification method.
Add 300 μl of Lysis buffer (8 M Ura, 2% Nonidet P-40, 2% ampholine (pH 3.5-10), 5% β-mercaptethanol, 5% polyvinylpyrrolidone) per 10 mg of this purified starch, and 100 degrees for 2 minutes. The heat treatment was performed with hot water. After ice-cooling for 10 minutes, the starch gelled solution was centrifuged at 15,000 rpm at 4 degrees for 10 minutes, and 200 μl of the supernatant was subjected to two-dimensional electrophoresis. For the first dimension electrophoresis, IEF consisting of 3.5% acrylamide gel and SDS-PAGE consisting of 15% bisacrylamide gel in the second dimension were performed.
The result of granular starch synthase detection is shown in FIG. Granular starch synthase-A1, B1, and D1 were detected at the positions shown in FIG. 1A, respectively, but no band was detected from the type (vii) wheat line (FIG. 1B). Thus, type (vii) wheat is completely deficient in granular starch synthase.

For the confirmation of starch synthase type II, a method already developed by the present inventors (Japanese Patent Application No. 2004-153904) was used. The method is as follows. Seeds were germinated and genomic DNA from young leaves was obtained using DNeasy plant mini kit manufactured by Qiagen. 100 mg of germinated seed seeds were taken and ground in liquid nitrogen until powdered. The ground sample was transferred to a 1.5 ml tube, and then buffer AP1 and RNase solution attached to the kit were added and heated at 65 ° C. for 10 minutes. Next, buffer AP2 was added and allowed to stand on ice for 5 minutes, and then the deposited precipitate was removed by centrifugation. After adding the buffer AP3 to the supernatant and mixing, the whole amount was subjected to a mini spin column and centrifuged to adsorb the DNA to the membrane. After washing with buffer AW twice, buffer AE was added and allowed to stand for 5 minutes, and the DNA solution was collected by centrifugation.
Sequence mutations that occurred on the gene sequences encoding the three starch synthase types II were confirmed by PCR. The primer sequences used are as follows.
Starch synthase type II-A1 SSIIAF1: GCGTTTACCCCACAGAGC (SEQ ID NO: 13)
SSIIAR1: ACGCGCCATACAGCAAGTCATA (SEQ ID NO: 17)
Starch synthase type II-B1 SSIIBF1: ATTTCTTCGGTACACCATTGGCTA (SEQ ID NO: 20)
SSIIBR1: TGCCGCAGCATGCC (SEQ ID NO: 23)
Starch synthase type II-D1 SSIIBF1: GGGAGCTGAAATTTTATTGCTTATTG (SEQ ID NO: 24)
SSIIBR1: TCGCGGTGAAGAGAACATGG (SEQ ID NO: 26)
The PCR reaction solution was prepared as follows. LA Taq (Takara Bio Inc.) was used for the reaction.
A primer manufactured by FASMAC was used.
GeneAmp PCR System 9700 (Applied Biosystems) was used as the PCR amplification apparatus, and the reaction conditions were set as follows.
The PCR amplification reaction solution was subjected to electrophoresis on a 3% agarose gel, followed by ethidium bromide staining, and the presence or absence of wild-type and mutant genes in the sample was confirmed by confirming DNA amplification bands of optimal size with each primer. Judged. FIG. 2 shows the results of genotyping of the type (i) control variety and the type (vii) wheat. Type (vii) indicates that all three starch synthase type II genes are mutated. From the above, it can be seen that type (vii) wheat is completely deficient in granular starch synthase and starch synthase type II.

(Confirmation of deficiency of starch synthase type II and granular starch synthase by SDS-PAGE)
Expression of starch synthase type II protein and granular starch synthase protein in each sample was confirmed by SDS-PAGE. 14 μl of SDS buffer per 1 mg of starch purified from type (i), type (ii), type (iii) and type (vii) was added and boiled for 5 minutes, and then ice-cooled for 2 minutes. After centrifugation at 15,000 rpm, 4 ° C. for 5 minutes, the supernatant was recovered and used as a sample for SDS-PAGE. For SDS-PAGE, an acrylamide gel prepared with an acrylamide solution mixed so that the ratio of acrylamide and methylenebisacrylamide was 30: 0.135 to a final concentration of 12.5% was used. It was. A silver staining kit (Daiichi Kagaku) was used to detect the protein after electrophoresis.
From this result, it was confirmed that type (vii) does not have all of starch synthase type II and granular starch synthase protein (FIG. 13).

2. The proportion of starch that does not form starch granules with a particle size of 10 μm or more in the accumulated starch When calculating the proportion of starch that does not form starch granules with a particle size of 10 μm or more in the accumulated starch, in the purification of the starch, the SDS buffer After washing with water and water, the gel-like layer and the lowermost starch grain layer were separated with a spatula. Each fraction was transferred to a pre-weighed 1.5 ml tube and the wet weight was measured. After drying the sample by natural drying, the dry weight was measured. For normal types of starch, the starch granule layer is almost 100% and a gel-like layer is hardly formed. In contrast, the dry weight of the starch grain layer derived from type (vii) wheat according to the present invention was 22 mg, whereas the dry weight of the gel-like layer was 13 mg. Therefore, the weight of the gel-like layer in the total weight of the collected starch fraction was about 37%.

3. Electron Microscope Observation An electron micrograph of seeds produced in a type (vii) wheat line is shown in FIG. Mature wheat seeds were roughly crushed, and the fracture surface was observed with an electron microscope (Keyence). Compared to type (i) wild-type starch, type (vii) starch granules are distorted, few in number, and mostly composed of smooth base material.

4). Chain length distribution analysis 4.1
The results of chain length distribution analysis of glucose branched chains constituting amylopectin contained in type (i), type (ii), type (iii) and type (vii) starch are shown in FIGS. The method is as follows. The purified starch was weighed, and 60 μL of 250 mM NaOH per 1 mg of starch was added and boiled for 20 minutes to complete gelatinization. After cooling, add 1.9 μL of acetic acid, 240 μL of 50 mM sodium acetate buffer (pH 4.0), 3.75 μL of 2% sodium azide, add 1 μL of Isoamylase (Nacalai), and react for 16 hours with stirring at 37 ° C. It was. After completion of the reaction, the enzyme was inactivated by boiling for 20 minutes, and then the supernatant was obtained by centrifugation at 14000 rpm, 20 ° C. for 2 minutes, and deionization was performed by adding a resin (manufactured by Bio-Rad). . After the resin was precipitated by centrifugation, the supernatant was transferred to a new tube, and the oligosaccharide concentration produced by the enzyme treatment was changed to the Park-Johnson method (Biochemical Experiment 19 “Starch and Related Sugar Experiments” Nakamura It was quantified by the Moral and Kainuma Junji Society Publishing Center, P123). Dilute the sample solution appropriately and add 50 μl of 25 μl of 0.1% potassium ferricyanide and 25 μl of potassium cyanide solution (0.48 g Na ₂ CO ₃ , 0.92 g NaHCO ₃ , 0.065 g KCN in water). , 100 ml) was added, sealed and heated in boiling water for 15 minutes. Then, cool on ice for 10 minutes, add 125 μl of 0.3% iron alum solution (1.5 g Fe · NH ₄ (SO ₄ ) ₂ · H ₂ O dissolved in 500 ml of 50 mM sulfuric acid) and let stand at room temperature for 20 minutes After that, the absorbance at 715 nm was measured. A calibration curve was created from the values measured by performing the same treatment on the glucose solution with the concentration varied between 0 and 10 mM, and the reducing end groups in the sample solution were quantified using this calibration curve. An amount equivalent to 5 nmol of each sample was collected and vacuum-centrifuged to prepare a sample for chain length distribution analysis. For the analysis of chain length distribution, a PA sugar chain analysis kit manufactured by Beckman was used. 2 μl of maltose quantitative control / mobility marker was added to the dried sample, and vacuum centrifugal drying was performed again. 2 μL of 1M Sodium Cyanoborohydride solution was added to the dried sample, and APTS labeling reagent was further added. The reaction was performed at 37 ° C. in the dark for 4 hours, and 46 μL of distilled water was added to stop the reaction. Furthermore, 5 μL was taken from this sample and diluted 40 times with distilled water to obtain a measurement sample. Beckman capillary electrophoresis apparatus PACEsystem 5000 was used for separation and quantification of oligosaccharides having different degrees of glucose polymerization. An eCAP N-CHO capillary was used as a separation capillary, and a sample was injected at a high pressure for 5 seconds, followed by electrophoresis at 30 kV for 30 minutes using an eCAP sugar analysis gel buffer. From the obtained peak chart, the peak areas of oligosaccharides having a degree of glucose polymerization of 3 or more and 70 or less were calculated, and the ratio of the area occupied by each peak was determined with the total peak area being 100%. The pattern obtained from the type (i) wheat line was used as a standard, and the value obtained by subtracting the proportion of the corresponding peak of type (i) from the proportion of the corresponding peak of each sample was graphed. In type (vii), the proportion of glucose side chains having a polymerization degree of 3 to 10 is remarkably increased compared to type (i) which is a standard strain, and conversely, the ratio of glucose side chains having a polymerization degree of 11 to 24 is increased. It has fallen (FIG. 4).
This is clearly different from the pattern of type (ii) (FIG. 5) or type (iii) (FIG. 6) which is a parent strain. In particular, the sugar component, which could not take the starch granule structure, constituted a smooth base material due to a decrease in the proportion of glucose side chains having an amylose content of 1% or less and a polymerization degree of 3 to 5. Conceivable.

4.2
The embryo was removed from the seeds, ground with a pestle and mortar, and then transferred to a 1.5 ml tube. 50 μl of 100% DMSO was added per 1 mg of ground seed weight. The solution was sealed and boiled for 30 minutes to complete gelatinization, and insoluble components were removed by centrifugation. 2 μl of 0.5 M sodium acetate buffer (pH 4.0) and 7 units of isoamylase (Nacalai) were added to 20 μl of the obtained sample solution, and the volume was made up to 100 μl with water (Reaction 1).
At the same time, by processing the same sample with isoamylase that has been deactivated by boiling in advance in this reaction solution, a sample for measuring the free oligosaccharide contained in the sample in advance (Reaction 2). After reacting at 37 ° C for 16 hours or longer, boil for 5 minutes to inactivate isoamylase, and calculate the concentration of the glucose branch chain generated by the reaction for reaction 1 using the modified Park-Johnson method (described above). A solution amount corresponding to 10 nmol was vacuum-centrifuge dried. For reaction 2, the same amount of solution as obtained in reaction 1 was dried. Thereafter, 2 μl of 1M Sodium Cyanoborohydride (in THF) solution was added, and further 2 μl of APTS labeling reagent was added, and the reaction was performed in the dark at 60 ° C. for 90 minutes. The reaction was stopped by adding 46 μl of water, and further diluted 40 times with water to obtain a sample for measurement. Analysis using PACEsystem 5000 was performed in the same manner as described above. By subtracting the area value of the corresponding peak obtained from Reaction 2 from the peak area value of each glucose branched chain obtained from Reaction 1, the value of the glucose branched chain produced by the isoamylase treatment was calculated. The area value was calculated for branched chains having a degree of polymerization of 1 or more and 60 or less, and the ratio of the area occupied by each peak was determined with the total as 100%.
Compared with type (i), type (vii) clearly had an increased proportion of branched chains having a degree of polymerization of 2 to 5 (FIGS. 7 and 8). In FIG. 4 of the example, the branched chain having a polymerization degree of 3 or more is analyzed, but in this example, the analysis was performed without using a maltose quantitative control / mobility marker that overlaps the peak of the polymerization degree 2. The peak area of the branched chain of degree 2 was also revealed. As a result, in type (vii), the peak with a degree of polymerization of 2 was significantly higher than in type (i).
Moreover, type (ii) amylopectin has almost the same structure as type (i) (FIG. 5), and there is almost no change in the chain length structure of amylopectin. On the other hand, in the same sticky types (vi) and (x) as in type (ii) (FIG. 12), the proportion of branched chains having a degree of polymerization of about 2 to 10 increased compared to type (ii). From 25 to 25 chains are lowered. This indicates that there is a change in the amylopectin structure, and as a result, the gelatinization peak temperature in DSC was lowered, and the solution viscosity after gelatinization was considered to be lowered.

5. Method for measuring amylose content The amylose content in starch of each wheat line was measured. The measurement method is as follows. Starch was gelatinized by adding 25 μl of ethanol per 1 mg of purified starch, adding 225 μl of 1M sodium hydroxide solution and heating in boiling water for 10 minutes. This gelatinization solution was diluted 10-fold with water, and 50 μl was collected for measurement. To this, 10 μl of 1M acetic acid, 20 μl of iodine solution, and 920 μl of water were added and mixed well. After leaving at 27 ° C. for 20 minutes, the absorbance at 620 nm was measured. Separately, potato-derived amylose was used as a standard curve preparation sample, and waxy corn-derived starch (starch containing no amylose) was used as a blank for the same treatment. The amylose content of each sample was measured by subtracting the absorbance value determined from waxy corn from the absorbance of each sample and applying it to the calibration curve data.

6). Degradation of raw starch with α-amylase The digestibility of raw starch by digestion with α-amylase treatment was examined as follows. The method of implementation was carried out by an improved method referring to “Starch / Related Sugar Experimental Method, P189-P192”. Raw starches derived from types (i), (ii), (iii), and (vii) of wheat lines were weighed and adjusted to a 1% suspension with water. This suspension was divided into three 20 μL suspensions (I, II, and III), and 230 μL of 0.8 M acetic acid buffer (pH 6.0) was added to I and II. III was added with 10 μL of 2M NaOH, and then heated at 50 ° C. for 5 minutes to obtain a completely gelatinized starch sample. After neutralizing with 20 μL of 1M acetic acid, 200 μL of 0.8M acetic acid buffer (pH 6.0) was added. Thereafter, 10 U of α-Amylase (manufactured by Megazyme) was added to I and III. In II, an equal amount of a solution inactivated by boiling the same α-Amylase for 20 minutes was added. All samples were reacted at 40 ° C. with stirring. After 12 hours, the reaction was stopped by diluting the reaction solution 5-fold with water and storing at 4 ° C. Oligosaccharides produced by α-Amylase treatment were quantified by a modification of the Park-Johnson method. The following formula yielded the raw starch degradation rate.
(I-II) / (III-II) * 100

7). Gelatinization degree After gelatinization by heating, the degree of white turbidity and the gelatinization degree when the solution was cooled to room temperature were examined. The method was performed as follows with reference to “Starch / Related Sugar Experimental Method, P189 to P192”. Purified starches of types (i), (iii), (iv) and (v) were weighed and adjusted to a 1% suspension with water. This suspension was divided into three 20 μL suspensions (I, II, III), and I and II were boiled for 20 minutes and cooled to room temperature. The degree of cloudiness of each sample at this time was confirmed visually. Then, after storing at 4 ° C. for 2 hours, 230 μL of 0.8 M acetic acid buffer (pH 6.0) was added. III was added with 10 μL of 2M NaOH, boiled for 5 minutes, cooled to room temperature, neutralized with 20 μL of 1M acetic acid, and then added with 200 μL of 0.8M acetate buffer (pH 6.0). Thereafter, 5 μL of β-Amylase (manufactured by SIGMA) and Pullulanase (manufactured by Hayashibara Biochemical Laboratories) were added to each of I and III. In II, an equal amount of a solution which was inactivated by boiling the same kind of enzyme for 20 minutes was added. All samples were reacted at 40 ° C. with stirring. After 30 minutes, the reaction was stopped by diluting the reaction solution 5-fold with water and storing at 4 ° C. Oligosaccharides produced by the enzyme treatment were quantified by a modification of the above Park-Johnson method. After calculating the oligosaccharide concentration of each sample, the gelatinization degree was calculated by the following formula.
(I-II) / (III-II) * 100

8). Viscosity After gelatinizing each wheat strain-derived starch, the viscosity of the solution when cooled to 60 degrees was measured. The measurement method is as follows. The purified starch was weighed and a 4% starch suspension was prepared with distilled water. Boiled in boiling water for 20 minutes and stored at 60 ° C. 100 μL of this paste solution was transferred to a vertically standing glass tube with an inner diameter of 1 mm, and the time required for the tip of the liquid surface to drop a distance of 5 cm was measured. On the other hand, various starch solutions prepared with waxy corn (manufactured by Toyotomi) from 7.5% to 0.5% are gelatinized and kept in the same manner, and the fall rate is measured in the same way to create a calibration curve. did. On the other hand, for waxy corn, the viscosity at 60 ° C. after gelatinization was measured with a rapid visco analyzer at the same concentration, and a calibration curve was prepared using this. Relative viscosity was calculated by fitting the falling speed at 60 ° C. of various wheat-derived starch solutions to a calibration curve prepared using waxy corn.

9. Freeze-thaw resistance after gelatinization Freeze-thaw resistance was examined as follows. Water was added to the purified starch of each sample to make a 5% starch suspension. After boiling for 20 minutes to gelatinize the starch suspension, it was cooled to room temperature, and first the state of the gelatinized solution at this point was observed. Samples that became cloudy and gelled by cooling to room temperature were evaluated as “high”, “medium”, and “low” depending on the degree. In addition, a sample in which neither cloudiness nor gelation was observed was evaluated as “no cloudiness”. These paste solutions or gelled samples were frozen at −80 ° C. for 1 hour and then thawed at 25 ° C. for 30 minutes, and then the state of the samples was observed. "X" for gelation at the end of the tenth melting, "△" for low gelation degree, and "○" for almost no gelation. Was evaluated.

The results of the above characterization for each type of wheat starch are summarized in Table 2 below.

As for type (vii), as described above, since the sugar component can no longer take a starch granule structure, it is easily affected by a degrading enzyme such as α-Amylase, and the degradability in raw starch is remarkable. This is thought to have increased. The type (vii) wheat line having such a sugar component as a main component can provide a completely different utilization method from the conventional method in terms of processability or digestibility when used as food. Specific examples include easily digestible bread, noodles, and confectionery. It may also be suitable as a feed crop. In addition, the amount of gelatinization energy during processing is very small compared to the system that forms starch granules. That is, cooking at a low temperature is possible, and the influence of heating on other food components can be minimized. In addition, since it is excellent in freeze-thaw resistance after gelatinization, it is particularly suitable for use in foods for refrigeration and frozen storage. In addition, type (vii) starch has a very high water absorption during starch purification compared to others. If the amount of water absorption is high, a large amount of water can be absorbed in a small amount when used as a gelling agent. Or when applying to a jelly-like food, preparation in a small amount is possible. It can also be applied to the production of resistant starch.
It is widely known that the content of amylose varies depending on the combination of granular starch synthase genes that synthesize it. On the other hand, it is known that when all starch synthase type II is deficient as described above, the amylose content is markedly increased compared to the wild type (type (iii)). From the above, if the combination of granular starch synthase is the same, the apparent amylose content will increase if one or two starch synthase types II are deleted at the same time. However, in practice, the apparent amylose content is almost the same value (type (iv) and type (ix), type (v) and type (viii) are compared)), or tends to decrease slightly. Found (data not shown).
In the examination of aging resistance (the higher the degree of cloudiness is, the lower the resistance), even if the type of granular starch synthase is the same, the resistance is increased when starch synthase type II is deleted at the same time. (Comparison between type (iv) and type (ix), type (v) and type (viii)), the relative viscosity of the paste after gelatinization was reduced (type (ii) and type (vi), Compare (vii)).
From the above, not only granular starch synthase, but also at least one starch synthase type II at the same time has an amylose content comparable to various conventional low amylose content wheat, and It was possible to produce wheat with improved aging resistance or with reduced viscosity of the paste after gelatinization. It was also found that this effect was more effective when two or more starch synthase types II were deleted. Low amylose wheat is widely used as a flour for noodles, and improvement of gelatinization and aging characteristics may have an improvement effect for such applications. In addition, it can be applied to other skins such as dumplings and buns.
In addition, type (vi) wheat according to the present invention is a type in which all of granular starch synthase is deleted and only one starch synthase type II is expressed, but compared to type (ii) wheat. The viscosity is significantly reduced. Type (ii) wheat is known to have a low viscosity of paste liquid when it is cooled after gelatinization, but type (vi) has a novel property of exhibiting a lower viscosity than that.
The above data indicate that it was possible to select wheat lines that accumulate starch of the type that could not be selected by selecting wheat lines based on the presence or absence of expression of granular starch synthase. .
In the present invention, hexaploid wheat having a novel starch is invented, but if the present invention is applied, tetraploid wheat having the same effect can be produced. It has long been known that tetraploid wheat can be obtained from the progeny of crosses of hexaploid and tetraploid wheat. Therefore, by crossing the wheat of the present invention that expresses granular starch synthase and starch synthase type II in a desired combination with tetraploid wheat, and combining the above selection methods from subsequent generations, granular starch synthase and starch synthesis Tetraploid wheat having enzyme type II in the desired combination can be selected. Examples of tetraploid wheat include durum wheat.

Ten. Measurement of maltose and glucose content The seed sample used for the measurement is lightly pulverized in advance separately from the sugar content measurement sample, dried at 135 ° C for 2 hours, and the moisture content is calculated in advance from the weight change before and after drying. deep.
Mature seeds, excluding type (i) and type (vii) wheat embryos, were ground in liquid nitrogen, mixed with 1 ml DMSO per 10 mg of ground powder, and allowed to stand at room temperature with occasional stirring. . After 1.5 hours, the mixture was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was transferred to a new tube. After boiling for 10 minutes, a certain amount was accurately measured and vacuum-dried (DMSO extracted sample).
In addition, dry seeds, excluding type (i) and type (vii) wheat embryos, were ground in liquid nitrogen, then added with 1 ml of 80% ethanol per 10 mg of flour and boiled for 20 minutes. It was. After cooling, the mixture was centrifuged at 10,000 rpm for 5 minutes, and a certain amount was accurately measured and vacuum-dried (ethanol extracted sample).
In the same way as described in the chain length distribution analysis, add 2 μl of 1M sodium cyanoborohydride solution and 2 μl of APTS labeling reagent, perform the reaction for 4 hours at 37 ° C. in the dark, and then add 46 μl of distilled water. The reaction was stopped. Furthermore, it diluted with distilled water 40 times, and was set as the sample for a measurement. The analyzer and conditions were measured under the same conditions as described in the chain length distribution analysis. Based on a calibration curve prepared in advance using a standard substance, the concentration was calculated from the peak areas of glucose and maltose, each sugar concentration per seed dry weight was calculated, and comparison between samples was performed.

11． Measurement of sucrose content Mature seeds excluding type (i) and type (vii) wheat embryos were ground in liquid nitrogen, then 1 ml of 80% ethanol was added per 10 mg of flour and the lid was sealed and boiled. I went for 20 minutes. After cooling, it was centrifuged at 10,000 rpm for 5 minutes, and a certain amount of the supernatant was accurately measured and vacuum-dried. Add sterilized water in the same volume as before drying to dissolve the sample, filter through a 0.45 μm filter, and filter the sample using an ultrafiltration membrane with a molecular weight cut off of 10,000. It was. The sucrose content in the sample was analyzed using a HP capillary electrophoresis apparatus manufactured by HewlettPackard, and a commercially available buffer (basic anion buffer for HPCE (Agilent)) and capillary (CE standard capillary (50 μm, 104 cm) (Agilent)) Separation was performed using The sucrose content in the samples was calculated in light of a calibration curve prepared in advance using commercially available sucrose, and the samples were compared.

(Results of sugar content measurement)
Type (vii) contains at least 5 times more glucose, at least 10 times more maltose, and at least 2 times more sucrose than type (i), which is the same type that is widely used commercially. It was recognized that The seeds of type (vii) contain these sugars up to a level where sweetness is felt even in the mouth, which can be said to be a novel feature not found in conventional types. The increase in the sugar content not only eliminates the need to add a new sugar component when processing the wheat of the present invention into food, but also enables use as a raw material for supplying the sugar itself.

12． Pasting properties (DSC)
About 3 mg of starch purified from each wheat was weighed, added with 3 times the amount of water, and hydrated for more than half a day. This sample is sealed in a sealed cell, and heated by a differential scanning calorimeter at a rate of 5 ° C./min from 30 ° C. to 105 ° C., gelatinization start temperature (T0), gelatinization peak temperature (Tp), Gelatinization end temperature (Tc) and enthalpy were measured.
As a result, type (ii), which is the parent line of type (vii), had an increase in T0 compared to type (i), which was the wild type, and decreased in type (iii), which was the other parent line. On the other hand, type (vii) showed a lower gelatinization peak temperature than type (iii), which is the parent strain. Nevertheless, the enthalpy is larger than that of type (iii), which is a very characteristic characteristic. Also, mochi-type starch (type (ii)) lacking all three granular starch synthases has a higher gelatinization peak temperature than type (i) with all three granular starch synthases. Widely known. In contrast, type (vi) has no amylose but has a lower gelatinization peak temperature than type (i). Not only the granular starch synthase but also two starch synthase types II were deleted simultaneously, so that the gelatinization peak temperature and enthalpy could be reduced.
When the gelatinization peak temperature in DSC measurement is lowered, gelatinization at a lower temperature becomes possible, which not only greatly affects workability but also enables processing with materials not suitable for heating. Furthermore, if gelatinization progresses at a lower temperature, it becomes more susceptible to degradation of amylase and the like, and the sugar released by these will lead to further sweetness, which may have a great influence on the taste.

13. Measurement of water-soluble polyglucan (WSP) content Mature seeds from which embryos had been removed were crushed and the fractions passed through a 65 μm nylon mesh were used. The collected fraction was boiled in methanol for 15 minutes and then centrifuged to collect a precipitate. The precipitate was further washed with 90% methanol, collected by centrifugation, dried and weighed. About 50 mg of the sample was weighed, 20 μl of water was added to 1 mg, and soluble polyglucan was extracted while stirring at room temperature for 20 minutes. The soluble fraction and the insoluble fraction were separated and collected by centrifugation at 600 × g for 20 minutes. Among these, the insoluble fraction was extracted again with water, and the soluble fraction and the insoluble fraction were recovered. The first and second soluble fractions were mixed and used for subsequent operations.
The mixed soluble fraction was added with trichloroacetic acid (TCA) to a final concentration of 5% for deproteinization and left on ice for 3.5 hours. The protein layer precipitated by centrifugation at 2,400 × g for 10 minutes was removed, and 3 times the amount of methanol was added to the supernatant to precipitate WSP, which was collected by centrifugation, dried and weighed.
The above water-insoluble fraction was suspended by adding 1050 μl of water, and then 150 μl of toluene was added, followed by stirring for 1 hour for protein removal treatment. The precipitate was recovered by centrifugation at 700 × g for 10 minutes. The protein removal treatment was further repeated twice, and the finally collected precipitate was washed with water three times and with methanol once, and then vacuum-dried and weighed. In each sample, the weight of the water-insoluble fraction and the weight of WSP were added to obtain the weight of the total sugar amount, and the ratio of the WSP weight to this was calculated (FIG. 14). The content of WSP was clearly increased in type (vii) versus Type (i), (ii), and (iii).

14. Bakery food For the production of bakery food, the wheat of the present invention type (vii) was used. The harvested mature seeds were mixed well by adding 1.5 ml of water per 100 g of seeds, tempering for 30 minutes, and then milled using a Brabender test mill Quadrumat Jr. to obtain wheat flour. The yield at this time was 46% by weight. Next, the bread shown in Table 4 and Table 5 and the following manufacturing process produced bread. In Table 4, the materials other than shortening were mixed and mixed with a mixer for 1 minute at low speed and for 2 minutes at high speed (27 ° C.). After the mixer was stopped and shortening was added, mixing was performed again for 1 minute at a low speed and for 3 minutes at a high speed, and the dough was fermented at 27 ° C. and 75% humidity for 90 minutes. After punching, it was again fermented under the same conditions for 30 minutes, divided into 100 g, rounded, and benched for 20 minutes. After shaping with a molder, it was baked by heating for 40 minutes in a fermentation chamber at 38 ° C and 85% humidity (205 ° C, 20 minutes).
The taste and texture of bread produced under the above conditions were evaluated by 10 panelists. Five-stage evaluation was performed according to the items listed in Table 6 and evaluation criteria, and an average value for each item was calculated. By summing up these average values, an overall evaluation of each test section was made (Table 7). From this result, it was confirmed that by using the flour obtained from the wheat of the present invention, a bakery food having a sweet taste and a strong flavor such as aroma and taste could be produced. Moreover, when manufacturing a bakery foodstuff, it turned out that it is preferable to use 0.1-60 mass parts, and it is more preferable to use 0.5-50 mass parts.

It is a schematic diagram of the electrophoresis result obtained by type (i) two-dimensional electrophoresis. It is a two-dimensional electrophoresis result of type (vii). It is the result of having performed the genotyping of the control cultivar of type (i) and the wheat of type (vii). It is an electron micrograph in seeds produced in a type (vii) wheat line. It is the chain length distribution analysis result ((vii)-(i)) of the glucose side chain which comprises amylopectin in the starch derived from a type (vii) system | strain. It is the chain length distribution analysis result ((ii)-(i)) of the glucose side chain which comprises amylopectin in type (ii) strain-derived starch. It is the chain length distribution analysis result ((iii)-(i)) of the glucose side chain which comprises amylopectin in type (iii) type | system | group origin starch. It is the chain length distribution analysis result of the glucose side chain which comprises amylopectin in the starch derived from a type (i) system | strain. It is the chain length distribution analysis result of the glucose side chain which comprises amylopectin in the starch derived from a type (vii) type | system | group. It is a graph which shows the glucose content in a mature seed. It is a graph which shows the maltose content in a mature seed. It is a graph which shows the sucrose content in a mature seed. It is the chain length distribution analysis result of the glucose side chain which comprises amylopectin in the starch derived from a type (x) system | strain. It is the electrophoresis result by SDS-PAGE. It is a graph which shows the soluble polyglucan content in a mature seed.

Claims (5)

Wheat that does not express all of the following proteins:
(1) Starch synthase type II-A1 protein encoded by the starch synthase type II-A1 gene of SEQ ID NO: 1,
(2) Starch synthase type II-B1 protein encoded by the starch synthase type II-B1 gene of SEQ ID NO: 3,
(3) Starch synthase type II-D1 protein encoded by the starch synthase type II-D1 gene of SEQ ID NO: 5,
(4) Granular starch synthase-A1 protein encoded by the granular starch synthase-A1 gene of SEQ ID NO: 7,
(5) Granular starch synthase-B1 protein encoded by the granular starch synthase-B1 gene of SEQ ID NO: 9, and (6) Granularity encoded by the granular starch synthase-D1 gene of SEQ ID NO: 11 Starch synthase-D1 protein. Wheat that does not have all the enzyme activities of the following proteins:
(1) Starch synthase type II-A1 protein encoded by the starch synthase type II-A1 gene of SEQ ID NO: 1,
(2) Starch synthase type II-B1 protein encoded by the starch synthase type II-B1 gene of SEQ ID NO: 3,
(3) Starch synthase type II-D1 protein encoded by the starch synthase type II-D1 gene of SEQ ID NO: 5,
(4) Granular starch synthase-A1 protein encoded by the granular starch synthase-A1 gene of SEQ ID NO: 7,
(5) Granular starch synthase-B1 protein encoded by the granular starch synthase-B1 gene of SEQ ID NO: 9, and (6) Granularity encoded by the granular starch synthase-D1 gene of SEQ ID NO: 11 Starch synthase-D1 protein. A food comprising wheat-derived flour according to claim 1 or 2 . The food according to claim 3 , wherein the food is a bakery food. A product processed using wheat-derived flour according to claim 1 or 2 .